We examined the signaling pathways regulating glycogen synthase (GS) in primary cultures of rat hepatocytes. The activation of GS by insulin and glucose was completely reversed by the phosphatidylinositol 3-kinase inhibitor wortmannin. Wortmannin also inhibited insulin-induced phosphorylation and activation of protein kinase B/Akt (PKB/Akt) as well as insulin-induced inactivation of GS kinase-3 (GSK-3), consistent with a role for the phosphatidylinositol 3-kinase/PKB-Akt/ GSK-3 axis in insulin-induced GS activation. Although wortmannin completely inhibited the significantly greater level of GS activation produced by the insulinmimetic bisperoxovanadium 1,10-phenanthroline (bpV-(phen)), there was only minimal accompanying inhibition of bpV(phen)-induced phosphorylation and activation of PKB/Akt, and inactivation of GSK-3. Thus, PKB/Akt activation and GSK-3 inactivation may be necessary but are not sufficient to induce GS activation in rat hepatocytes. Rapamycin partially inhibited the GS activation induced by bpV(phen) but not that effected by insulin. Both insulin-and bpV(phen)-induced activation of the atypical protein kinase C (/) (PKC (/)) was reversed by wortmannin. Inhibition of PKC (/) with a pseudosubstrate peptide had no effect on GS activation by insulin, but substantially reversed GS activation by bpV(phen). The combination of this inhibitor with rapamycin produced an additive inhibitory effect on bpV-(phen)-mediated GS activation. Taken together, our results indicate that the signaling components mammalian target of rapamycin and PKC (/) as well as other yet to be defined effector(s) contribute to the modulation of GS in rat hepatocytes.Much current work has focused on defining the nature of the downstream effectors mediating key effects of insulin. The regulation of glycogen synthase (GS), 1 the rate-limiting enzyme in glycogen synthesis, by insulin has received increasing attention. GS is regulated by a complex interplay of diurnal, nutritional, and hormonal factors (reviewed in Refs. 1-3) that ultimately modulate the phosphorylation state and hence the activity of the enzyme. Whereas there have been numerous studies of GS activation in skeletal muscle, the modulators of GS in liver have been less completely evaluated. Studies to date indicate that glucose and insulin are the major physiologic effectors of hepatic GS activation (1, 2). It appears that glucose and/or its metabolite glucose 6-phosphate activate hepatic GS by physically associating with critical regulatory enzymes upstream of GS or with GS itself (2, 4, 5), although other mechanisms have been suggested (6). The mechanisms by which insulin effects activation of GS and stimulation of glycogen synthesis in liver are poorly defined, although data have been obtained indicating that modulation of PP1-G (1, 7) as well as PKB/Akt (8) and GSK-3 (9) may be involved. Insulin activation of the insulin receptor kinase (IRK) is followed by tyrosine phosphorylation of insulin receptor substrates (IRSs), the two major ones in liver being IRS-1 and -2 ...

The hormonal regulation of insulin-like growth factor binding protein (IGFBP)-1 and -4 mRNA was compared in serum-free primary rat hepatocyte cultures. The combination of dexamethasone and glucagon (Dex/Gluc) strongly increased IGFBP-1 and IGFBP-4 mRNA levels. Insulin suppressed Dex/Gluc-stimulated IGFBP-1 but not IGFBP-4 mRNA levels. In contrast, the peroxovanadium compound, bisperoxovanadium 1,10-phenanthroline (bpV(phen)), completely abrogated Dex/Gluc induction of both IGFBP mRNA species. Wortmannin and rapamycin blocked the inhibitory effect of insulin but not that of bpV(phen) on Dex/Gluc-stimulated IGFBP mRNA. Thus, although phosphatidylinositol 3'-kinase and p70s6k are necessary for insulin-mediated transcriptional inhibition of the IGFBP-1 gene, a signaling pathway, independent of phosphatidyloinositol 3'-kinase and p70s6k, is activated by bpV(phen) and mediates IGFBP-1 as well as IGFBP-4 mRNA inhibition. Mitogen-activated protein (MAP) kinase activity induced by insulin was suppressed to below basal levels in the presence of Dex/Gluc, whereas in response to bpV(phen), MAP kinase activity was high and unaffected by Dex/Gluc, consistent with a role of MAP kinases in bpV(phen)-mediated inhibition of IGFBP mRNA. The specific MAP kinase kinase (MEK) inhibitor, PD98059, inhibited insulin but not bpV(phen)-stimulated MAP kinase activity, suggesting that MAP kinases can be activated in a MEK-independent fashion. Peroxovanadium compounds are strong inhibitors of tyrosine phosphatases, which may inhibit specific tyrosine/threonine phosphatases involved in the negative regulation of MAP kinases.

Peroxovanadiums (pVs) are potent protein tyrosine phosphatase (PTP) inhibitors with insulin-mimetic properties in vivo and in vitro. We have established the existence of an insulin receptor kinase (IRK)-associated PTP whose inhibition by pVs correlates closely with IRK tyrosine phosphorylation, activation, and downstream signaling. pVs have also been shown to activate various tyrosine kinases (TKs) that could participate in activation of the insulin-signaling pathway. In the present study we have sought to determine whether pV-induced IRK tyrosine phosphorylation requires the intrinsic kinase activity of the IRK, and whether IRK activation is necessary to realize the early steps in the insulin-signaling cascade. To address this we evaluated the effect of a pure pV compound, bis peroxovanadium 1,10-phenanthroline [bpV(phen)], in HTC rat hepatoma cells overexpressing normal (HTC-IR) or kinase-deficient (HTC-M1030) mutant IRKs. We showed that at a dose of 0.1 mM, but not 1 mM, bpV(phen) induced IRK-dependent events. Thus, 0.1 mM bpV(phen) increased tyrosine phosphorylation and IRK activity in HTC-IR but not HTC-M1030 cells. Tyrosine phosphorylation of insulin signal-transducing molecules was promoted in HTC-IR but not HTC-M1030 cells by bpV(phen). The association of p185 and p60 with the src homology-2 (SH2) domains of Syp and the p85-regulatory subunit of phosphatidylinositol 3'-kinase was induced by bpV(phen) in HTC-IR, but not in HTC-M1030 cells, as was insulin receptor substrate-1-associated phosphatidylinositol 3'-kinase activity. Thus autophosphorylation and activation of the IRK by bpV(phen) is effected by the IRK itself, and the early events in the insulin- signaling cascade follow from this activation event. This establishes a critical role for PTP(s) in the regulation of IRK activity. bpV(phen) could be distinguished from insulin only in its ability to activate ERK1 in HTC-M1030 cells, thus indicating that this event is IRK independent, consistent with our previous hypothesis that bpV(phen) inhibits a PTP involved in the negative regulation of mitogen-activated protein kinases.

The mitogenic response to insulin and epidermal growth factor (EGF) was studied in subconfluent and confluent cultures of primary rat hepatocytes. In subconfluent cultures, wortmannin, LY294002, and rapamycin reversed insulin- and EGF-induced [3H]thymidine incorporation into DNA. The mitogen-activated protein kinase (MAPK) kinase 1 (MEK1) inhibitor PD98059 was without significant effect on either insulin- or EGF-induced [3H]thymidine incorporation. Insulin treatment did not alter levels of messenger RNAs (mRNAs) for c-fos, c-jun, and c-myc. EGF induced an increase in c-myc, but not c-fos or c-jun, mRNA levels in subconfluent hepatocyte cultures. This increase in c-myc mRNA was abolished by PD98059. In confluent cells that could not be induced to synthesize DNA, EGF treatment also promoted an increase in c-myc mRNA to levels seen in subconfluent cultures. This increase was also abrogated by PD98059. These data indicate that in primary rat hepatocyte cultures, 1) the phosphoinositol 3-kinase pathway, perhaps through p70s6k activation, regulates DNA synthesis in response to insulin and EGF; 2) the MAPKpathway is not involved in insulin- and EGF-induced DNA synthesis; and 3) p44/42 MAPKs are involved the induction of c-myc mRNA levels, although this induction is not required for DNA synthesis. These studies define two distinct signal transduction pathways that independently mediate growth-related responses in a physiologically relevant, normal cell system.

The authors compared, in a double-blind, randomized, crossover study in 13 healthy adult volunteers, the single- and multiple-dose pharmacokinetics, relative bioavailability, and side effects of a new oral sustained-release formulation of codeine (SRC) containing 150 mg codeine base, with oral immediate-release codeine phosphate (IRC). Sustained-release codeine was given at a dose of 150 mg every 12 hours for 5 doses; IRC was given at a dose of 60 mg (2 x 30 mg) every 4 hours for the first 3 doses, and 30 mg every 4 hours thereafter for 12 doses. Plasma codeine levels were determined using a sensitive and specific high-performance liquid chromatography method and corrected for dose administered and codeine base equivalent. Mean values for single-dose pharmacokinetic parameters for SRC and IRC, respectively, were: Cmax of 217.8 and 138.8 ng/mL; Tmax of 2.3 and 1.1 hours; AUC0-inf of 1202.3 and 1262.4 ng.mL-1.hour-1; and t1/2el of 2.6 hours for both formulations. Their respective mean steady-state pharmacokinetic parameters were: Cmax of 263.8 and 222.9 ng/mL; Tmax of 3.2 and 1.1 hours; AUC0-12h of 1576.4 and 1379.1 ng.mL-1.hour-1; and t1/2el of 2.8 and 2.3 hours. These results indicate comparable bioavailability between both formulations with SRC providing delayed peak plasma levels. The sustained-release character of SRC can be explained by a delayed absorption, which is not limiting to drug elimination. Sustained-release codeine provides higher plasma codeine levels over a broader time interval and is expected to improve pain management.